Ce travail est soutenu en partie par le National Institutes of Health Grant GM-26738 (à la JAT).

1. PAK2 activation <ol><li> Ajouter la pré-testés appropriée le montant de la caspase 3 à 20 ul de 12 mg / ml dans le tampon A PAK2 et (50 mM DTT mM Tris-HCl, 150 mM de NaCl, et 2 à pH 7,5) incuber à 34 ° C pendant 30 min pleinement cliver et activer PAK2. </li><li> Analyser les produits de clivage par SDS-PAGE (10%) et Coommassie coloration au bleu. </li><li> Ajouter le PAK2 clivé pour l&#39;activation de tampon B (50 mM Tris-HCl, 150 mM NaCl, 15 mM MgCl2, 10 mM ATP) à pH 7,5, dans 20 ul d&#39;une concentration finale de 10 mg / ml PAK2, et incuber à 34 ° C pendant 30 min pour activer complètement PAK2 par autophosphorylation sur 8 sites. </li><li> Confirmer le clivage complet de PAK2 par la migration de l&#39;p34 et p27 fragments tel que déterminé par SDS-PAGE 10. Vérification adéquate des conditions autophosphorylation se fait via autoradiographie dans une expérience séparée en utilisant (32 P) l&#39;ATP ou la spectrométrie de masse pour détecter phosphopeptides. </li></ol> 2. Identificationde la pepsine à digérer PAK2 Fragments <ol><li> Ajouter 1 ml d&#39;acide trifluoroacétique à 0,1% (TFA) à pH 2,5 à laver les billes d&#39;agarose deux fois avant d&#39;utiliser. Puis, centrifuger les échantillons pendant 15 s à 2000 xg pour éliminer les bourrelets. </li><li> Ajouter PAK2 (20 pg dans 2 pi de NaCl 150 mM, DTT 50 mM Tris-HCl et 2 à pH 7,5) à 100 ul de 0,1% de TFA, et incuber l&#39;échantillon pendant 5 min avec 30 ul de billes d&#39;agarose pepsine conjugués . </li><li> Equilibrer une HPLC en phase inverse C18 avec un système de solvant primaire de TFA 0,1% à pH 2,5. La digestion par la pepsine (50 pi) est élue en utilisant un linéaire de 100 min gradient de 0 - 80% d&#39;acétonitrile contenant 0,1% de TFA à un débit de 0,2 ml / min. Les fractions sont collectées toutes les 2 min. </li><li> Sécher les fractions HPLC avec un speed-vac (3 h) et remettre en suspension les échantillons dans une solution d&#39;acétonitrile (5 pi) et de 0,1% de TFA (5 pi) à pH 2,5. </li><li> Initialement, chaque fraction est projeté pour les peptides à l&#39;aide du MALDI-TOF Voyager DE PerSeptive Biosystems STR (PE biosystems, Foster City, Californie, Etats-Unis). </li><li> Les 16 fractions contenant les peptides sont soumis à une spectrométrie de masse en tandem en utilisant un spectromètre de masse Q-TOF et un QSTAR XL oMALDI MS / MS pour identifier les fragments. </li><li> La séquence de chaque peptide est vérifiée par les ions produits générés par la spectrométrie de masse tandem avec l&#39;théoriques m / z ratios basés sur la séquence primaire de PAK2 utilisant le site Web de protéines Prospector ( <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. Mesure de l&#39;amide échange H / D <ol><li> Tenir toutes les solutions et les réactifs nécessaires pour échange H / D à 25 ° C dans un bain d&#39;eau. L&#39;ATP est dissous dans l&#39;eau et le pH est ajusté à 7,0 avant utilisation. L&#39;ATP 4 mM supplémentaires dans le tampon D 2 O est de prévenir l&#39;ATP dissociée de PAK2 après dilution. </li><li> Initier échange H / D par addition de 18 pl d&#39;tamponné D 2 O (50 mM MOPS pH 6,98, 125 mM de NaCl) à 2 ul (20 pg) De PAK2 dans un tampon contenant 150 mM de NaCl, 50 mM DTT mM Tris-HCl (pH 7,5) et 2, résultant en un niveau PAK2 de 10 mg / ml et un pH de 7,0 à 0 ° C. </li><li> Incuber les échantillons en trois exemplaires, l&#39;échange H / D de 0, 0,5, 1, 3, 5 et 10 min, ou 24 h. </li><li> Quench le processus échange H / D en ajoutant 180 pl d&#39;glacée TFA 0,1% à pH 2,2, ce qui amène le pH à 2,5 dans un volume final de 200 pl. Pour les échantillons contenant de l&#39;ATP, 180 pl d&#39;glacée TFA 0,11% à pH 2,2 est utilisé pour maintenir le pH à 2,5 à l&#39;état trempé. </li><li> Ajouter une aliquote (100 pi) de chaque échantillon trempé à 30 ul de billes d&#39;agarose activé la pepsine-conjugué. La digestion par la pepsine est autorisé à procéder sur la glace pendant cinq minutes et les échantillons sont vortexés toutes les 30 secondes. </li><li> Mettre fin à la digestion par centrifugation de l&#39;échantillon pendant 15 s à 5000 xg à 4 ° C pour éliminer la pepsine. </li><li> Rapidement congeler les échantillons dans l&#39;azote liquide 2 et conserver à -80 ° C pendant plus de 2jours avant analyse MALDI-TOF. </li><li> La matrice pour MALDI est de 5 mg / ml d&#39;α-cyano-4-hydroxycinnamique (ACSSD) dissous dans une solution (1:1:1) de l&#39;acétonitrile, l&#39;éthanol et 0,1% de TFA à pH 2,5 et maintenue à 0 ° C. </li><li> Décongeler partiellement chaque échantillon rapidement et mélanger 1 ul de chaque échantillon avec 1 pl de matrice et place sur une plaque à 4 ° C cible MALDI. </li><li> Appliquer un vide modéré à la plaque à sécher les taches en 30 secondes avant analyse MALDI-TOF. Le temps écoulé entre la décongélation de l&#39;échantillon et extraction du spectre est d&#39;environ 3 min. </li><li> Acquérir des spectres de masse MALDI-TOF avec un STR PerSeptive Biosystems Voyager DE. Acquérir des données à un taux d&#39;échantillonnage de 2 GHz à 100 000 canaux de données, avec une tension de 20.000 V accélération, et une tension de grille de 65% en utilisant l&#39;extraction retardée avec un retard d&#39;impulsion de 180 ns. Une centaine de scans sont en moyenne en 2 min. L&#39;instrument MALDI-TOF n&#39;est pas limité à n&#39;importe quelle marque ou tout autre logiciel particulier. </li><li> Utilisez le programme Data Explorer 4.0 à l&#39;ordinateur Calculmangé la deutération de chaque fragment. </li><li> Calculer l&#39;échange de retour basée sur le ratio du nombre deutéron incorporé réel à 24 heures de échange H / D divisé par tous les sites possibles échangeables, qui sont les amides épine dorsale, sauf l&#39;amide N-terminale (figure 1). </li></ol> 4. Les résultats représentatifs: Le clivage des caspases et l&#39;autophosphorylation sont vérifiées par SDS-PAGE coloration Commassie. PAK2 inactif est une seule bande de 58 kDa. Le clivage de la caspase PAK2 est complet, et produit deux fragments, p27 et p34 qui migrent séparément. La migration de p27 et p34 autophosphorylée montre un chevauchement sur ​​le gel en raison de la migration de l&#39;retardé totalement phosphorylés p27 fragment (Figure 2). H / D des expériences de change sont effectuées plusieurs fois entre 0 et 10 min (figure 3). À titre d&#39;exemple, le cours moment de l&#39;incorporation de deutérium dans le m / zpEAK 1697.85 des PAK2 inactif est composé de plusieurs pics isotopiques comme le montre l&#39;évolution de l&#39;enveloppe de masse au cours du temps. <img alt="Figure 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> Figure 1. Équation pour le calcul de la deutération. <img alt="Figure 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> Figure 2. Autophosphorylation et le clivage des caspases d&#39;PAK2. Caspase-clivé PAK2 (voie de gauche), intactes PAK2 inactifs (au milieu voies) et de la caspase-clivé PAK2 autophosphorylée (voie de droite) ont été analysés par SDS-PAGE et coloration au bleu de Coommassie. Adapté de Hsu, AJ et al 2. <img alt="Figure 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> Figure 3. Spectre de masse d&#39;un cours moment de l&#39;incorporation de deutérium pendant PAK2 inactif. PAK2 inactifs a été soumis à H / D ExchaESN pour les 0-10 min et analysés par MALDI-TOF. Un exemple de la distribution élargie isotopique de MALDI-TOF du pic m / z 1697,85 au cours du temps d&#39;échange H / D est montrée. La ligne pointillée rouge indique la moyenne de l&#39;enveloppe de masse et le déplacement de l&#39;enveloppe de masse montre l&#39;deutération d&#39;un peptide.

<ol>
	<li>Anand, G. S., Hughes, C. A., Jones, J. M., Taylor, S. S., Komives, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amide+H/2H+exchange+reveals+communication+between+the+cAMP+and+catalytic+subunit-binding+sites+in+the+R(I)alpha+subunit+of+protein+kinase.">Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase.</a> A. J. Mol. Biol. 323, 377-386 (2002).</li><li>Hsu, Y. H., Johnson, D. A., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+conformational+changes+during+activation+of+protein+kinase+Pak2+by+amide+hydrogen/deuterium+exchange.">Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.</a> J. Biol. Chem. 283, 36397-36405 (2008).</li><li>Englander, S. W., Kallenbach, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+exchange+and+structural+dynamics+of+proteins+and+nucleic+acids.">Hydrogen exchange and structural dynamics of proteins and nucleic acids.</a> Q. Rev. Biophys. 16, 521-655 (1983).</li><li>Katta, V., Chait, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+changes+in+proteins+probed+by+hydrogen-exchange+electrospray-ionization+mass+spectrometry.">Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry.</a> Rapid Commun. Mass. Spectrom. 5, 214-217 (1991).</li><li>Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localizing+the+membrane+binding+region+of+Group+VIA+Ca2+-independent+phospholipase+A2+using+peptide+amide+hydrogen/deuterium+exchange+mass+spectrometry.">Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 284, 23652-23661 (2009).</li><li>Hsu, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+rigidifies+the+C2+domain+and+the+intradomain+interaction+of+GIVA+phospholipase+A2+as+revealed+by+hydrogen/deuterium+exchange+mass+spectrometry.">Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.</a> J. Biol. Chem. 283, 9820-9827 (2008).</li><li>Lee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+hPAK65+by+caspase+cleavage+induces+some+of+the+morphological+and+biochemical+changes+of+apoptosis.">Activation of hPAK65 by caspase cleavage induces some of the morphological and biochemical changes of apoptosis.</a> Proc. Natl. Acad. Sci. U. S. A. 94, 13642-13647 (1997).</li><li>Rudel, T., Bokoch, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+morphological+changes+in+apoptotic+cells+regulated+by+caspase-mediated+activation+of+PAK2.">Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2.</a> Science. 276, 1571-1574 (1997).</li><li>Roig, J., Traugh, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytostatic+p21+G+protein-activated+protein+kinase+gamma-PAK.">Cytostatic p21 G protein-activated protein kinase gamma-PAK.</a> Vitam. Horm. 62, 167-198 (2001).</li><li>Walter, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+and+activation+of+p21-activated+protein+kinase+gamma-PAK+by+CPP32+(caspase+3).+Effects+of+autophosphorylation+on+activity.">Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.</a> J. Biol. Chem. 273, 28733-28739 (1998).</li><li>Clauser, K. R., Baker, P., Burlingame, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+accurate+mass+measurement+(+/-+10+ppm)+in+protein+identification+strategies+employing+MS+or+MS/MS+and+database+searching.">Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.</a> Anal. Chem. 71, 2871-2882 (1999).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Identification de la pepsine fragments digérés est une étape critique de l&#39;expérience échange H / D. Identification incorrecte du peptide peut conduire à une conclusion erronée. PAK2 pepsine-digérée est éluée à travers une colonne HPLC phase inverse C18 pour obtenir un fond propre. Dans nos expériences, un total de 40 fractions ont été recueillies et soumises à MALDI-TOF. Peptides multiples pourraient être identifiés dans chacune des fractions. Les peptides ont été soumis à la spectrométrie de masse (Q-TOF MS / MS et oMALDI MS / MS) pour identifier leurs séquences. Les spectres MS / MS d&#39;un peptide devrait avoir au moins trois ions de produit pour confirmer l&#39;identification du peptide. Le programme Data Explorer 4.0 ordinateur (Applied Biosystems) effectue la correction de base initial et la filtration du bruit. Chaque spectre doit être étalonné sur deux pics séquencé. Nous calibrer notre spectre par la masse théorique de l&#39;undeuterated m / z qui sont 923,45 et 1697,84. TIl précision de la masse après l&#39;étalonnage peut atteindre 10 ppm. Après deutération, les mono-isotopique peut passer à un supérieur de masse. Augmentations d&#39;échelon unitaire de ces rapports m / z donné les masses améliorée associée à deutération. Les intensités maximales de l&#39;enveloppe de masse ne sera pas affecter le niveau de deutération. Toutefois, les intensités des pics des pics isotopiques dans une enveloppe de masse sont particulièrement critiques pour le calcul de la masse moyenne. La première étape du calcul de la deutéron est incorporé soustrayant la masse peptidique de l&#39;échantillon des non-deutérés de l&#39;échantillon deutérié. Trois autres facteurs, la D 2 O de dilution, les deutons résiduelle dans les chaînes latérales et l&#39;échange dos, devra être examiné plus avant dans le calcul (figure 1). La dilution de D 2 O est le facteur de dilution de D 2 O après le mélange de l&#39;échantillon et D 2 O buffer pour initier l&#39;échange H / D. Le deuton résiduelle (4,5%) dans les chaînes latérales de la pepsine digérerpeptides éd doit être soustraite. L&#39;arrière-échange est la perte inévitable de deutération dans tous les peptides deutérés et la pepsine-digérés dans le processus de mesure de masse. Le peptide le plus accessible au solvant (m / z = 1105,60) après 24 heures d&#39;échange H / D représente une région pleine de deutération. Le nombre deutéron constituée pour chacun des peptides est la différence entre le centroïde des peptides deutérés et non deutérés peptique PAK2. Le plus hautement deutéré peptide m / z 1105 a été choisie pour représenter deutération plein quand PAK2 était à 24 heures de échange H / D. Le ratio d&#39;échange Retour a été calculé par le nombre deutéron incorporé réel à 24 heures de échange H / D divisé par tous les sites possibles échangeables à peptide m / z 1105.

<table border="1"></table><table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> D 2 O </td><td> Aldrich </td><td> 7789-20-0 </td><td></td></tr><tr><td> α-cyano-4-hydroxycinnamique </td><td> Sigma </td><td> 28166-41-8 </td><td></td></tr><tr><td> MALDI-TOF </td><td> PE Biosystems </td><td> Voyager DE STR </td><td></td></tr><tr><td> Q-TOF spectromètre de masse </td><td> Q-TOF Ultima-Global </td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI MS / MS </td><td> Applied Biosystems </td><td></td><td></td></tr><tr><td> Data Explorer 4.0 programme informatique </td><td> Applied Biosystems </td><td></td><td></td></tr&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

Amide hydrogène / deutérium échange (échange H / D) couplée à la spectrométrie de masse a été largement utilisée pour analyser l&#39;interface des interactions protéine-protéine, protéine de conformation change, la dynamique des protéines et des interactions protéine-ligand. Échange H / D sur les positions amide épine dorsale a été utilisé pour mesurer le taux de deutération des micro-régions dans une protéine par spectrométrie de masse 1,2,3. La résolution de cette méthode dépend de digestion par la pepsine de la protéine deutérée d&#39;intérêt en peptides qui varient normalement de 3 à 20 résidus. Bien que la résolution de l&#39;échange H / D mesurée par spectrométrie de masse est inférieure à la résolution seul résidu mesuré par le simple hétéronucléaire cohérence quantique (HSQC) méthode de RMN, la spectrométrie de masse en mesure échange H / D n&#39;est pas limité par la taille de la protéines 4. Échange H / D est réalisé dans une solution aqueuse qui maintient la conformation des protéines. Nous fournissons une méthode qui utilIZES l&#39;MALDI-TOF pour la détection de deux, au lieu d&#39;un système HPLC / ESI (ionisation par électrospray)-MS 5,6. Le MALDI-TOF fournit des données précises sur l&#39;intensité de masse pour les peptides de la protéine digérée, dans ce cas la protéine kinase PAK2 (aussi appelée γ-Pak). Protéolyse de Pak 2 est réalisée en une digestion par la pepsine déconnecté. Cette méthode alternative, lorsque l&#39;utilisateur n&#39;a pas accès à une colonne de CLHP et la pepsine connecté à la spectrométrie de masse, ou lorsque la colonne de pepsine sur HPLC ne résulte pas en une carte de digestion optimale, par exemple, le disulfure fortement lié phospholipase A 2 sécrétée (SPLA 2). Utilisant cette méthode, nous avons réussi à surveiller l&#39;évolution du niveau lors de l&#39;activation de la deutération PAK2 par la caspase 3 clivage et l&#39;autophosphorylation 7,8,9.

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI-TOF spectrométrie de masse a été utilisé avec succès pour contrôler l&#39;amide d&#39;hydrogène / deutérium de change dans la protéine kinase PAK2 activation.

Amide hydrogène / deutérium Analyse Bourse &amp; MALDI-TOF spectrométrie de masse de PAK2 activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

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17.7K vues.  Tunghai University. MALDI-TOF spectrométrie de masse a été utilisé avec succès pour contrôler l'amide d'hydrogène / deutérium de change dans la protéine kinase PAK2 activation.

Vidéo: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

Préparation des échantillons MALDI: la méthode de la couche ultra mince

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

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Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

L&#39;analyse des complexes protéiques par spectrométrie de masse Grande structurels

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

L&#39;onde T de mobilité ionique et spectrométrie de masse: Procédures de base expérimentale pour l&#39;analyse de protéines complexes

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

Analyse directe des cellules isolées par spectrométrie de masse à la pression atmosphérique

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profilage thiol redox du protéome par spectrométrie de masse isotopique de l&#39;étiquetage

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

L&#39;utilisation de MALDI-TOF spectrométrie de masse et une base de données personnalisée pour caractériser les bactéries indigènes à un environnement unique Cave (Kartchner, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Selected Reaction Monitoring spectrométrie de masse pour Absolute quantification des protéines

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Enrichissement de matrice extracellulaire de protéines à partir de tissus et la digestion en peptides pour l&#39;analyse de spectrométrie de masse

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Applications de la mono-sonde: Mass Spectrometry Imaging et simple Cellule d&#39;analyse dans les conditions ambiantes

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flux de travail complet pour l&#39;analyse des histones modifications post-traductionnelles utilisant la spectrométrie de masse Bottom-up: De Histone Extraction à l&#39;analyse des données

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...